The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
Protein C (human precursor: Swiss-Prot P04070, Annotation Release 44, July 2004, which is hereby incorporated in its entirety herein) is a vitamin K-dependent serine protease involved in blood coagulation. Synthesized as a single chain precursor, protein C is cleaved into a light chain and a heavy chain connected by a disulfide bond. The “latent” form of the enzyme contains an “activation peptide” at the amino terminus of the heavy chain. Protein C is activated by thrombin/thrombomodulin-mediated cleavage of this peptide to produce active protein C (APC).
Protein C levels and/or function are affected by numerous pathological states, including inherited protein C deficiencies, activated protein C resistance, deficiencies of protein S, deficiencies of antithrombin, and acquired protein C deficiencies. The latter is caused by a variety of conditions, including disseminated intravascular coagulation, deep vein thrombosis, pulmonary embolism, and anticoagulant therapy. In systemic inflammatory response syndromes, the interplay of coagulation state and inflammation can result in decreased protein C activity, which has been reported to be of diagnostic and prognostic significance. Recombinantly produced human activated protein C (drotrecogin alfa, or XIGRIS® (Eli Lilly)) is the first drug approved by the U.S. F.D.A. for treatment of severe sepsis. See, e.g., Hosac, BUMC Proceedings 15: 224-227, 2002; Kinasewitz et al., Crit. Care 8: R82-R90, 2004; dePont et al., Crit. Care 9: R490-R497, 2005. Each of the foregoing is hereby incorporated in their entirety herein.
Assays for protein C generally fall into one of two classes: functional assays that measure the serine protease activity of active protein C, and immunological assays that detect total protein C. See, e.g., Axelsson, Protein C Product Monograph 1995, Chromogenix AB; Liaw et al., J. Thromb. Haemost. 1:662-70, 2003. Each of the foregoing is hereby incorporated in their entirety herein. These assays share certain common features: the ability to detect active forms of protein C, and a demonstrated relationship to diagnosis and prognosis of septic patients. In the presence of administered XIGRIS, however, such assays may not be reflective of the physiological state of the patient, as both XIGRIS and endogenous protein C will contribute to the assay signal obtained.
Antibodies that bind to protein C heavy chain but not to activated protein C have been described in the scientific literature. See, e.g., Takahashi et al., Biochim. Biophys. Acta. 1161:113-23, 1993; Vincenot et al., FEBS Lett. 432: 94-97, 1998. Each of the foregoing is hereby incorporated in their entirety herein. Although these antibodies have been reported to inhibit cleavage of the protein C heavy chain by thrombin/thrombomodulin, there is no report of immunoassays using such antibodies to detect latent protein C in patient samples, or that if such immunoassays were to be provided, such assays would be reflective of the physiological state of patients.